.gamma.-Aminobutyric acid (GABA) is the major inhibitory neurotransmitter in the mammalian central nervous system. The major type of receptor for the inhibitory neurotransmitter .gamma.-aminobutyric acid (GABA), called the GABA.sub.A receptor, is a member of a gene superfamily of ligand-gated ion channels. GABA, the endogenous ligand for the GABA.sub.A -complex, stimulates chloride ion conductance through the associated chloride ion channel. The predominant effect of GABA is the interaction with a specific receptor protein which results in an increase of the chloride ion conductance of the post-synaptic membrane to produce an inhibition of neuronal firing.
The GABA.sub.A receptor is a multi-subunit ligand-gated ion channel. This receptor is a heterooligomeric protein composed of several distinct polypeptide types. Four different classes of subunit have been defined; .alpha., .beta., .gamma., and .delta.. Multiple variants within these classes exist. Sequences of seven .alpha., three .beta., two .gamma. and one .delta. subunits have been reported. Molecular cloning of these polypeptides reveals that they show 20-40% identity with each other, and 10-20% identity with polypeptides of the nicotinic acetylcholine receptors and strychnine-sensitive glycine receptor. Each polypeptide type is also represented by a family of genes whose members have 60-80% amino acid sequence identity. Regions of conserved and variable amino acid sequence suggest structural and functional domains within each polypeptide. All of the polypeptides when expressed in heterologous cells produce GABA-activated chloride channels, and the different subtypes express different pharmacological properties. The distributions of mRNAs for the different GABA.sub.A receptor polypeptides and their subtypes show significant brain regional variation consistent with pharmacological and biochemical evidence for receptor heterogeneity. Subpopulations of GABA.sub.A receptors with different cellular and regional locations show differential sensitivity to GABA, to modulators like steroids, to physiological regulation, to disease processes, and to pharmacological manipulation by drugs such as benzodiazepines. The properties of the different subpopulations of GABA.sub.A receptors are determined by which of the one or more different polypeptides and their subtypes are expressed in a given cell to produce a variety of different oligomeric protein structures.
The GABA.sub.A -receptor chloride-ionophore complex (GABA.sub.A -complex) is the primary site of action for many of the drugs used to treat anxiety and seizure disorders such as the benzodiazepines and anticonvulsant barbiturates. By allosteric drug-induced modulation the receptors serve as molecular control elements through which the levels of anxiety, vigilance, muscle tension and epileptiform activity can be regulated. The allosteric modulation of the GABA.sub.A receptor by different agents acting at independent sites on the GABA.sub.A -complex can be demonstrated by a variety of methods including electrophysiological measurements with intact cells and isolated membrane fragments (e.g. patch clamp preparations), radioligand binding studies using tissue homogenates, and by .sup.36 Cl-flux measurements using tissue slices, cultured neurons, and cell free membrane vesicle preparations.
There is a need for a stable in vitro system useful to identify and characterize compounds that have GABA.sub.A receptor binding properties and activities similar to those of the known active benzodiazepine compounds. According to the present invention, stable permanent lines of transformed host cells are provided which contain GABA.sub.A receptors that bind to benzodiazepines. These cells lines can be useful to identify compounds with activities similar to those of the known benzodiazepines using patch clamp preparations as well as the other methods available to observe receptor activity. The stable permanent cell lines can be cultured to provide a consistent laboratory research model.